Actions of individual molecules come into focus

PORTLAND, Ore.  Nanoscale waveguides measuring as small as 40 nm have enabled Cornell University researchers to observe individual molecules during chemical reactions.

Using e-beam lithography to pattern a photoresist prior to a reactive ion etch, the researchers fabricated 2 million "holes" in an aluminum layer over a glass substrate. The tiny holes served both as waveguides for a 48-nanometer laser and as chemical-reaction chambers, enabling the group to observe the behavior of individual molecules as chemical reactions were under way.

"Its easy to dilute a solution enough to get a single molecule, but the resulting chemical reactions will not represent what happens in natural concentrations. Our zero-mode waveguide technique, on the other hand, [marks the first time] that anyone has been able to observe these chemical reactions in their natural concentrations," said postdoctoral associate Michael Levene, a member of professor Watt Webb's research group at Cornell (Ithaca, N.Y.). Levene performed the work along with professor Harold Craighead's research group, comprising postdoctoral associates Stephen Turner and Mathieu Foquet and graduate student Jonas Korlach.

Roughly 1,000 molecules occupy the volume defined by the wavelength of visible light, complicating attempts to resolve individual molecules and observe their behavior via optical microscopy techniques. A technique called near-field microscopy has reduced the observed volume of a sample to about 100 molecules, in their natural concentrations; but extreme dilution would still be required to get the sample down to a single molecule.

By contrast, the zero-mode technique, according to Levene, reduces the observed volume by 10,000 times, to just 2,500 cubic nanometers  100 times smaller than the best competing techniques.

A waveguide's size is usually of the same order as the light passing through it  its "cutoff" threshold wavelength  and any light of longer wavelength than the threshold is blocked. As a result, a waveguide that is smaller than the light impinging on it should pass nothing. That is why the Cornell group dubs its waveguide zero-mode: The 488-nm light should not pass through a 40-nm waveguide.

"In single-mode waveguides, there is only one way that light can go down through the guide, and once you are below the threshold, there are no allowed modes for the light," said Levene. "Most EEs assume that waveguides are only useful above their cutoff threshold; but here we have found an application where we make a very simple version of a waveguide  just a hole in a metal film  that's far below cutoff, and yet we found a very good use for it."

Nanoscale lab

The microchip was created at the Cornell Nanoscale Science and Technology Facility with funds from the National Science Foundation, the U.S. Department of Energy and the National Institutes of Health.

The university has licensed the technology to an Ithaca startup.

"There has already been some commercialization; two of the guys who worked on this project [Turner and Craighead] have started a company called Nanofluidics Inc.," said Levene. "Cornell owns all the patents, but people should contact Nanofluidics if they want to obtain zero-mode waveguides."

The reasoning that led to the experiment started with the observation that even though light cannot pass through the waveguides, because they are zero-mode, it nevertheless might penetrate a few nanometers into the hole. To verify that prediction, the researchers illuminated 40-nm holes with 488-nm wavelength light.

"Nature hates a discontinuity, so the light does go into the hole a little bit. We focused a 488-nm laser beam on an individual hole, and despite the fact that the hole is so small that the light cannot penetrate into it  we are so far below threshold  the light did penetrate about 10 to 15 nm," said Levene.

The resulting illuminated volume was so small that only one molecule or a few of them could fit inside, thereby simplifying the observations of individual molecular reactions.

"The walls of the waveguide provided lateral resolution, and the depth of resolution was determined because the light cannot penetrate very far," said Levene.

To prove that, the researcher filled the chip's holes with a solution containing DNA molecules that are approximately the same size as the holes' width, but since the holes were about 100 nm deep, they could not be sure that exactly one molecule was protruding into the illuminated volume. Consequently, their first test merely determined which holes had no molecules, a single molecule, or more than one.

Once they determined which holes had exactly one DNA molecule, they focused their detector on only those holes and recorded when a second chemical in the mix  a fluorescently tagged nucleotide  reacted with the DNA molecule. Because the specific DNA molecule used functions as a "copy machine," assembling a second, identical DNA molecule, the observations made it possible to identify the exact sequence in which the copy was made.

Fluorescence burst

"We saw a burst of fluorescence each time a neucleotide attached to the DNA molecule. We used an avalanche photodiode to detect the bursts of fluorescence," said Levene. "Essentially we were watching the manufacture of a single molecule of DNA, one base at a time."

In the experiment, the researchers arbitrarily divided the 20 million holes into 25 groups of 90,000, but only because it was convenient to use a commercially available plastic gasket that happened to have 25 openings in it. For real work, in the future, Levene claimed that many better methods exist for isolating different chemicals in one or a few holes, enabling many different "tests" to be performed simultaneously.

"This technique is open to a massive amount of parallelism, but our intention in this experiment was just to prove the concept," said Levene.

The main research of the Webb group is DNA sequencing, so the massive parallelism will be used in that case to discover the structural sequences of bases in previously unknown samples of DNA. By tagging each of the four DNA bases with different fluorescent colors, each neucleotide can be observed as it is added to the copy.

To date, DNA sequencing has been limited to about 800 base pairs on a strand of DNA, but the zero-mode waveguide, according to Levene, enables decoding continuous sequences of hundreds of thousands of base pairs at a time. Likewise, the massive parallelism could be used to test thousands of drug candidates simultaneously.

"This technique has broad applicability to single-molecule enzymology in general," said Levene. A big advantage of the technique is the ability to create millions of parallel single-molecule reaction chambers on a single chip. The technology could be scaled up easily and interface with current commercial automated drug discovery systems, the researcher said.

Levene said the technique could also be used in other industries, such as finding new atomically accurate materials.

"We only illuminate the entrance to the hole, because the intensity of the light does rapidly decay as it goes inside," he said. " But in terms of optical efficiency, our method is much better than conventional near-field techniques, which lose three orders of magnitude or more of light going through the tiny holes. We use the light as it is decaying into an aperture, whereas conventional near-field approaches have to get the light all the way through an aperture."

Being able to observe the chemical behavior of single molecules could resolve some important issues in biochemistry because there is some variation in the time taken for a given reaction. Observations and theory are based on the average reaction times of large groups of molecules, so this technique should be able to add information to models of biochemical reactions.

(a) A laser beam is first expanded by a telescope (L1 and L2), then focused by a high-NA objective lens (OBJ) on a fluorescent sample (S). The epi-fluorescence is collected by
the same objective, reflected by a dichroic mirror (DM), focused by a tube lens (TL), filtered (F), and passed through a confocal aperture
(P) onto the detector (DET).

(b) Magnified focal volume (green) within which the sample particles (black circles) are illuminated. The focal volume is the distribution of laser illumination at the focus of the
objective. On the other hand, the observation volume, contained within the focal volume, is the region in space where fluorescent molecules are both excited and detected.

(c) A typical fluorescence signal, as a function of time, measured for rhodamine green (RG) with excitation wavelength lx=488 nm.

(d) Portion of same signal in (c), binned, with expanded time axis and average fluorescence Fbar. The signal F(t) at time t is correlated with itself at a later time (t+t) to produce the autocorrelation G(t).

(e) Measured G(t) describing the fluorescence fluctuation of RG molecules due to diffusion only as observed by FCS. Assuming a Gaussian observation volume, G(t) can be least-squares fitted using various analytic functions to extract information about molecular concentration, brightness, diffusion, and chemical kinetics, for one or more diffusing fluorescent species.